1. Field of the Invention
This invention relates to biologically active polypeptides based on transforming growth factor-beta sequences that have transforming growth factor-beta-like activity. In particular, this invention relates to the development of drugs for treatment of immune or inflammatory disorders, such as allograft rejection, arthritis, cancer, and viral infections, and to immunodiagnostic reagents.
2. Description of Related Art
The transforming growth factor-.beta. (TGF-.beta.) molecules identified thus far are each dimers containing two identical 112 residue polypeptide chains linked by disulfide bonds. The molecular mass of these dimers is about 25 kd. Biologically active TGF-.beta. has been defined as a molecule capable of inducing anchorage independent growth of target cell lines or rat fibroblasts in in vitro cell culture, when added together with EGF or TGF-.alpha. as a cofactor. TGF-.beta. is secreted by virtually all cell types in an inactive form. This latent form can be activated by proteolytic cleavage of mature TGF-.beta. from its precursor (at the Arg-Ala bond in position 278). A non-covalent complex is formed from the association of the mature TGF-.beta. with the precursor remainder or with a protein binding to TGF-.beta. or with alpha.sub.2 -macroglobulin. This complex is disrupted so as to activate the TGF-.beta. either by exposure to transient acidification or by the action of exogenous proteases such as plasmin or plasminogen activator.
There are at least three forms of TGF-.beta. currently identified, TGF-.beta..sub.1, TGF-.beta..sub.2, and TGF-.beta..sub.3. Suitable methods are known for purifying this family of TGF-.beta.s from various species such as human, mouse, green monkey, pig, bovine, and chick, and from various body sources such as bone, platelets, or placenta, for producing it in recombinant cell culture, and for determining its activity. See, for example, R. Derynck et al., Nature, 316:701-705 (1985); U.S. Ser. Nos. 715,142; 500,832; 500,833, all abandoned; European Pat. Pub. Nos. 200,341 published Dec. 10, 1986, 169,016 published Jan. 22, 1986, 268,561 published May 25, 1988, and 267,463 published May 18, 1988; G.B. Pat. Appln. 2,146,335 published Apr. 17, 1985; U.S Pat. No. 4,774,322; Seyedin et al., J. Biol. Chem., 262: 1946-1949 (1987); Cheifetz et al, Cell, 48: 409-415 (1987); Jakowlew et al., Molecular Endocrin., 2: 747-755 (1988); Dijke et al., Proc. Natl. Acad. Sci. (U.S.A.), 85: 4715-4719 (1988); Derynck et al., J. Biol Chem., 261: 4377-4379 (1986); Sharples et al., DNA, 6: 239-244 (1987); Derynck et al., Nucl. Acids. Res., 15: 3188-3189 (1987); Derynck et al., Nucl. Acids. Res., 15: 3187 (1987); Seyedin et al., J. Biol Chem., 261: 5693-5695 (1986); Jakowlew et al., Mol. Endrocrin., 2: 1186-1195 (1988); Madisen et al., DNA, 7: 1-8 (1988); and Hanks et al., Proc Natl. Acad. Sci. (U.S.A.), 85: 79-82 (1988), the entire contents of these publications being expressly incorporated by reference.
TGF-.beta. has been shown to have numerous regulatory actions on a wide variety of both normal and neoplastic cells. Recent studies indicate an important role for TGF-.beta. in cells of the immune system (J. Kehrl et al., J. Exp. Med. 163:1037 [1986]; H-J Ristow, Proc. Natl. Acad. Sci. U.S.A., 83:5531 [1986]; A. Rook et al., J. Immunol., 136:3916[1986]) and in proliferation of connective and soft tissue for wound healing applications (M. Sporn et al., Science, 219:1329 [1983]; R. Ignotz et al., J. Biol. Chem., 261:4337 [1986]; J. Varga et al., B.B. Res. Comm., 138:974 [1986]; A. Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 78:5339 [1981]; A. Roberts et al., Fed. Proc., 42:2621 [1983]; A. Roberts et al., Proc. Natl. Acad. Sci. U.S.A., 83:4167 [1986]; U.S. Ser. No. 500,833, supra; U.S. Pat. No. 4,774,228 to Seyedin et al.), as well as epithelia (T. Matsui et al., Proc. Natl. Acad. Sci. U.S.A., 83:2438 [1986]; G. Shipley et al. Cancer Res. 46:2068 [1986]). Moreover, TGF-.beta. has been described as a suppressor of cytokine (e.g., IFN-.gamma., TNF-.alpha.) production, indicating its use as an immunosuppressant for treating inflammatory disorders (Espevik et al., J. Exp. Med. 166: 571-576 [1987]; European Pat. Pub. No. 269,408 published June 1, 1988; U.S. Pat. No. 4,806,523 issued Feb. 21, 1989), and as a promoter of cachexia (Beutler and Cerami, New Eng. J. Med., 316: 379 [1987]). Further, TGF-.beta. induces collagen secretion in human fibroblast cultures (Roberts et al., Proc. Nat. Acad. Sci. USA 83: 4167-4171 (1986); Chua et al., J. Biol. Chem., 260:5213-5216 [1983]); stimulates the release of prostaglandins and mobilization of calcium (A Tashjian et al., Proc. Natl. Acad. Sci. U.S.A . 82:4535 [1985]); and inhibits endothelial regeneration (R. Heimark et al., Science 233:1078 [1986] ).
TGF-.beta. is multifunctional, as it can either stimulate or inhibit cell proliferation, differentiation, and other critical processes in cell function (M. Sporn, Science. 233:532 [1986]).
The multifunctional activity of TGF-.beta. is modulated by the influence of other growth factors present together with the TGF-.beta.. TGF-.beta. can function as either an inhibitor or an enhancer of anchorage-independent growth, depending on the particular set of growth factors, e.g., EGF or TGF-.alpha., operant in the cell together with TGF-.beta. (Roberts et al., Proc. Natl. Acad. Sci. U.S A., 82:119 [1985]). TGF-.beta. also can act in concert with EGF to cause proliferation and piling up of normal (but not rheumatoid) synovial cells (Brinkerhoff et al., Arthritis and Rheumatism. 26:1370 [1983]).
Most recently, TGF-.beta. has been found to suppress the expression of Class II histocompatibility antigens on human cells induced by human interferon-.gamma. (Czarniecki et al., J Immunol., 140: 4217-42223 [1988]; Czarniecki et al. J. Interferon Res., 7: 699 [1987]; Palladino et al., J. Cell. Biochem. Supp. 11A [Jan. 17-Feb. 5, 1987]. UCLA Symposia on Molecular and Cellular Biology, Alan R. Liss, Inc., New York, abstract A016. p. 10; Chiu et al., Triennial Symposium: Biology of Growth Factors, University of Toronot, Ontario, Canada, [June 17-19, 1987]; Palladino et al., Immunobiology, 175: 42 [1987]).
For a general review of TGF-.beta. and its actions, see Sporn et al., J. Cell Biol., 105: 1039-1045 (1987) and Sporn and Roberts, Nature, 332: 217-219 (1988).
Fragments of TGF-.beta. are described in EP 290,012 published 11/9/88 and EP 267,463 published 5/18/88. The former describes fragments of TGF-.beta.2 having at least about eight amino acids, for example, in the region of N-terminal amino acids 1 to 20, particularly 4-15, and more particularly 9-14, a C-terminal sequence, or a truncated N-terminal or C-terminal molecule. The latter patent publication describes in claim 36 a 20-mer consisting of an internal sequence of TGF-.beta.3.
TGF-.alpha. polypeptides that compete with the EGF receptor are disclosed in U.S. Pat. No. 4,816,561 issued Mar. 28, 1989.
A putatively hydrophilic region of murine retroviral envelope protein p15E has been identified that inhibits stimulated T-lymphocyte proliferation. Schmidt et al., Proc. Natl. Acad. Sci. U.S.A., 84: 7290-7294 (1987). U.S. Pat. No 4,822,606 issued Apr. 18, 1989 to R. Snyderman et al. discloses a class of immunosuppressive peptides that are based on a 26-amino acid sequence, or part thereof, that is conserved among a variety of retroviruses associated with immunosuppression, including murine and feline leukemia virus, human retrovirus, and a simian virus that causes AIDS in monkeys. One of these peptides (designated CKS-17) was synthesized to correspond to the conserved region of p15E and tested for its effect on a variety of immune cell functions. In addition, peptides corresponding to the conserved regions of the human retroviruses HTLV-I, -II, and -III were synthesized and similarly tested. These peptides have the formula: EQU A-Gln-B-Arg-C-D-E-F-G-H-I-J-K-L-M-N-O,
wherein:
A is Leu, Ala, or Tyr; PA0 B is Asn or Ala; PA0 C is Arg, Leu, or Ile; PA0 D is Gly, Ala, or Leu; PA0 E is Leu or Ala; PA0 F is Asp or Val; PA0 G is Leu, Tyr, Glu, or Ile; PA0 H is Leu or Arg; PA0 I is Phe, Leu, Tyr, or Thr; PA0 J is Leu, Trp, or Ala; PA0 K is Lys, Glu, Gln, or Ala; PA0 L is Glu, Gln, or Asp; PA0 M is Gly or Gln; PA0 N is Gly or Gln; and PA0 O is Leu, Val, or Ile. PA0 A is Val, Leu, Ala, or Tyr or a polypeptide that has at its C-terminus any one of the amino acids Val, Leu, Ala, or Tyr, and that, if greater than 15 residues does not have the sequence of mature or precursor TGF-.beta. at a homologous location in the mature or precursor TGF-.beta. molecule; PA0 B is Leu, Asn, Ala, Pro, Arg, or Gln; PA0 C is Arg or Leu; PA0 D is Arg, Leu, Ile, or Tyr; PA0 E is Gly, Ala, Leu, Ile; PA0 F is Asp, Leu, or Ala; PA0 G is Asp, Val, or Phe; PA0 H is Arg, Lys, Leu, Tyr, Glu, or Ile; PA0 I is Lys, Arg, Gln, or Leu; PA0 J is Asp, Leu, Phe, Tyr, or Thr; and PA0 K is Leu, Trp, or Ala, or a polypeptide that has at its N-terminus any one of the amino acids Leu, Trp, or Ala, and that, if greater than 15 residues, does not have the sequence of mature or precursor TGF-.beta. at a homologous location in the mature or precursor TGF-.beta. molecule; and physiologically acceptable salts or esters thereof; provided, however, that the polypeptide excludes (a) a full-length mature TGF-.beta. molecule or precursor TGF-.beta. molecule or deletion variants of mature or precursor TGF-.beta. molecules in which from about 1 to 10 amino acid residues have been deleted (b) a polypeptide of the sequence Cys-Val-Arg-Gln-Leu-Tyr-Ile-Asp-Phe-Arg-Lys-Asp-Leu-Gly-Trp-Lys, and (c) a polypeptide of the sequence: Arg-Asn-Leu-Glu-Glu-Asn-Cys-Cys-Val-Arg-Pro-Leu-Tyr-Ile-Asp-Phe-Arg-Gln-As p-Leu. PA0 L is Gly, Leu, Glu, Gln, or Ala; PA0 M is Trp, Glu, Gln, or Asp; and PA0 N is Lys, Gly, or Gln.
Immune modulators have applications in various fields involving the immune system. For example, they are useful in preventing graft rejection, preventing autoimmunity, or alleviating inflammatory reactions mediated by immune disorders. Immune modulators typically have suffered from numerous undesirable and substantial side effects.
Accordingly, it is an object of the present invention to provide immune modulators having transforming growth factor-beta-like activity without suffering from undesirable side effects.
It is another object to provide a novel class of immune modulators that are useful as immunogens to elicit antibodies to TGF-.beta., i.e., TGF-.beta. antagonists.
It is another object to provide a novel class of immune modulators that are useful to develop diagnostic assays for the presence in patient fluids of immunosuppressive proteins such as TGF-.beta., or antibodies to such proteins.
These and other objects will become apparent to those skilled in the art.